Gram-negative bacterial bloodstream infections and their attendant endotoxemia are associated with vascular leak syndromes, including the life-threatening, Acute Respiratory Distress Syndrome (ARDS). One receptor for endotoxin or bacterial lipopolysaccharide (LPS), Toll-like receptor (TLR)-4, is expressed by endothelial cell (EC)s. Although much is known about TLR4 signaling, how it is coupled to opening of the pulmonary microvascular endothelial paracellular pathway is unknown. We have demonstrated that LPS increases tyrosine phosphorylation of EC proteins enriched to EC-EC boundaries and induces actin reorganization. Prior PTK inhibition and F-actin stabilization each protects against LPS-induced barrier disruption. We now present data identifying the zonula adherens (ZA) proteins, VE-cadherin, 3-catenin, and p120ctn, as substrates for LPS- induced tyrosine phosphorylation, and activation of one or more src family PTK (SFK)s as a prerequisite for LPS-induced opening of the paracellular pathway. We propose the following SPECIFIC AIMS: 1. To identify the TLR4-responsive, MyD88-dependent signaling/adapter molecule(s) that couple the LPS stimulus to SFK activation and opening of the paracellular pathway in human lung microvascular endothelia (HMVEC-Ls). 2. To define LPS-induced changes in protein-protein interactions within the ZA multiprotein complex, the ZA-actin cytoskeletal linkage, and between opposing VE-cadherin ectodomains. 3. To identify the SFK(s) through which LPS increases tyrosine phosphorylation of junctional and/or actin cytoskeletal proteins, induces ZA reorganization, and opens the endothelial paracellular pathway. 4. To establish whether PTP[unreadable] counter-regulates SFK activation, increased tyrosine phosphorylation of ZA proteins, reorganization of the ZA multiprotein complex, and/or opening of the paracellular pathway, in response to the LPS stimulus. Understanding the mechanism(s) through which LPS regulates EC-EC homophilic adhesion and the paracellular pathway has implications not only for sepsis-associated acute lung injury and the movement of intravascular fluid and macromolecules into tissues but for neutrophil extravasation as well. PUBLIC HEALTH RELEVANCE: Numerous bacteria that cause life-threatening infections in humans, release a substance called endotoxin, which can bind to a receptor on the cell that line blood vessels. When endotoxin engages this receptor, it can trigger opening of the spaces between these cells and leakiness of body fluids into the tissues, including the air-spaces of the lung. Understanding the biochemical events that lead to this high-mortality, vascular leak syndrome should offer potential targets for therapeutic interventions.